Both of the organisms to be studied in this proposal, V. cholerae and ETEC, are significant causes of morbidity and mortality due to diarrheal illness in less economically developed countries leading to hundreds of thousands of deaths each year. Additionally, each has been implicated in outbreaks in recent years that have been addressed with genomic techniques. These outbreaks have highlighted the utility of genome sequencing in the analysis of the dissemination, evolution and pathogenesis of these pathogens. We will take advantage of two ongoing clinical trials at the Center for Vaccine Development on the University of Maryland campus to examine the host:pathogen interactions during a challenge with fully virulent V. cholerae or ETEC. We will be examining this interaction at the level of pathogen signaling and response, human immune response, and interactions with the existing microbiota. Small animal models for enteric diseases are not truly representative of the disease process that occurs in humans, so the ability to directly study the host and pathogen response within human subjects in a controlled manner is a unique opportunity and will provide many novel insights into these interactions. This will be the first time, to our knowledge that an enteric pathogen will be examined for the genetic variation that occurs and/or is selected for during transit through humans. Additionally, by using the most cutting edge 'omic technologies to examine the microbiota, metagenome and metatranscriptome, as well as immunological studies, we will be able to begin to model the interaction during the infectious process. In addition to the studies within the human host, we extend the examination of the transcriptional interactions to the use of an organoid model system. Since we realize that the human challenge experiments are rare, we hope to develop this organoid system as a more representative surrogate model for infection studies. We increase the complexity of the model system by coculturing the pathogens with other pathogens that have been identified to be isolated together in diarrheal studies, as well as we will extend the microbiota studies to co-culture the species/genera identified with an increased severity of disease in the challenge studies. These studies will provide an unprecedented view into the interaction of the host, pathogen and resident microbiota.